U.S. patent number 6,372,461 [Application Number 09/397,720] was granted by the patent office on 2002-04-16 for synthesis of vanillin from a carbon source.
This patent grant is currently assigned to Board of Directors operating Michigan State University. Invention is credited to John W. Frost.
United States Patent |
6,372,461 |
Frost |
April 16, 2002 |
**Please see images for:
( Certificate of Correction ) ** |
Synthesis of vanillin from a carbon source
Abstract
A bioengineered synthesis scheme for the production of vanillin
from a carbon source is provided. The bioconversion methods of the
present invention comprise the steps of microbe-catalyzed
conversion of a carbon source to vanillic acid followed by
enzyme-catalyzed reduction of the vanillic acid to produce
vanillin. As shown in the synthesis scheme of FIG. 2, the
microbe-catalyzed conversion step of the present invention requires
five enzymes which are provided by a recombinant microbe. In a
preferred embodiment, the recombinant microbe is Escherichia coli
designed to cause dehydration of 3-dehydroshikimic acid and
regioselective methylation of the resulting protocatechuic acid.
The enzyme-catalyzed reduction step of the present invention
comprises the reduction of vanillic acid to vanillin by
aryl-aldehyde dehydrogenase.
Inventors: |
Frost; John W. (Okemos,
MI) |
Assignee: |
Board of Directors operating
Michigan State University (East Lansing, MI)
|
Family
ID: |
22282303 |
Appl.
No.: |
09/397,720 |
Filed: |
September 16, 1999 |
Current U.S.
Class: |
435/156;
435/252.33; 435/320.1 |
Current CPC
Class: |
C12P
7/24 (20130101); C12N 9/0008 (20130101); C12N
15/52 (20130101) |
Current International
Class: |
C12P
7/24 (20060101); C12N 9/02 (20060101); C12N
015/00 (); C12N 001/20 () |
Field of
Search: |
;435/156,320.1,252.33 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Campbell, C.J. et al., "The End of Cheap Oil," Sci. Am.
278(3):77-83 (1998). .
Clark, G.S., "Vanillin," Perfum. Flavor. 15:45-54 (1990). .
Coward, J.K. et al., "Kinetic Studies on Catechol
O-Methyltransferase. Product Inhibition and the Nature of the
Catechol Binding Site," Biochemistry 12(12):2291-2297 (1973). .
Esposito, L. et al., "Vanillin," Kirk-Othmer Encyclopedia of
Chemical Technology, Fourth Ed., Kroschwitz, J.I., Howe-Grant, M.,
Ed., Wiley: New York, vol. 24:812-925 (1997). .
Falconnier, B. et al., "Vanillin as a product of ferulic acid
biotransformation by the white-rot fungus Pycnoporus cinabarinus
I-937: Identification of metabolic pathways," J. Biotechnol.
37:123-132 (1994). .
Gross, G.G. et al., "Reduction of Cinnamic Acid to Cinnamaldehyde
and Alcohol," Biochem. Biophy. Res. Commun. 32(2):173-178 (1968).
.
Gross, G.G. et al., "Reduktion aromatischer Sauren zu Aldehyden und
Alkoholen im zellfreien System," Eur. J. Biochem. 8:413-419 (1969).
.
Gross, G.G., "Formation and Reduction of Intermediate Acyladenylate
by Acryl-Aldehyde," Eur. J. Biochem. 31:585-592 (1972). .
Lesage-Meessen, L. et al., "A two-step bioconversion process for
vanillin production from ferulic acid combining Aspergillus niger
and Pycnoporus cinnabarinus," J. Biotechnol. 50:107-113 (1996).
.
Lesage-Meessen, L. et al., "An attemp to channel the transformation
of vanillic acid into vanillin by controlling methoxyhydroquinone
formation in Pycnoporus cinnabarinus with cellobiose," Appl.
Microbiol. Biotechnol. 47:393-397 (1997). .
Ranadive, A.S., "Vanilla--Cultivation, Curing, Chemistry,
Technology and Commercial Products," In Spices, Herbs, and Edible
Fungi, Charalambous, G., Ed., Elsevier: Amsterdam, pp. 517-577
(1994). .
Snell, K. et al., "Synthetic Modification of the Escherichia coli
Chromosome: Enhancing the Biocatalytic Conversion of Glucose into
Aromatic Chemicals," J. Am. Chem. Soc. 118(24):5605-5614 (1996).
.
Westcott, R.J. et al., "Use of Organized Viable Vanilla Plant
Aerial Roots for the Production of Natural Vanillin,"
Phytochemistry 35(1):135-138 (1994). .
Zenk, M.H. et al., "The Enzymic Reduction of Cinnamic Acids,"
Recent Adv. Phytochem. 4:87-106 (1972). .
Tuomainen et al. "Validation of assay of
catechol-O-methyltransferase activity in human erythrocytes",
(1996), J Pharm Biomed Anal 14:515-523.* .
Weaver et al. "Cloning of an aroF allele encoding a tyrosine
insensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase",
(1990) J Bacteriol 172:6581-6584.* .
Guldberg, H.A. et al., "Catechol-O-Methyl Transferase:
Pharmacological Aspects and Physiological Role," Pharmacological
Reviews 27(2):135-206 (1975). .
Reenila, I. et al., "Opposite Effect of Ethanol on Recombinant
Membrane-Bound and Soluble Activities of
Catechol-O-methyltransferase," Pharmacology & Toxicology
77:414-416 (1995)..
|
Primary Examiner: Prouty; Rebecca E.
Assistant Examiner: Steadman; David
Attorney, Agent or Firm: Harness, Dickey & Pierce,
P.L.C.
Government Interests
Work on this invention was sponsored in part by the United States
Department Of Agriculture Grant No. 95-37500-1930 and the National
Science Foundation Grant No. CHE963368 amendment 002. The
Government may have certain rights in the invention.
Parent Case Text
This application claims benefit of provisional application No.
60/100,937, filed Sep. 18, 1998.
Claims
What is claimed is:
1. A method of synthesizing vanillin from a carbon source
compsing:
a) converting the carbon source to vanillic acid with a microbe
comprising a recombinant gene encoding a 3-dehydroshikimate
dehydratase and a recomnbinant gene encoding
catechol-O-methyltransferase; and
b) reducing the vanillic acid to vanillin with aryl-aldelhyde
dehydrogenase.
2. The method of claim 1, wherein the gene encoding
3-dehydroshikimate dehydratase is the aroZ gene.
3. The method of claim 1, wherein the gene encoding
catechol-O-methyltransferase is the P.sub.tac COMT gene.
4. The method of claim 3, wherein the P.sub.tac COMT gene is
located on a plasmid in the microbe.
5. The method of claim 4, wherein the plasmid is pKL5.97A.
6. The method of claim 1, wherein the microbe is E. coli
KL7/pKL5.97A identified by ATCC designation anmber 98859.
7. The method of claim 1, wherein the microbe further comprises a
gene encoding for an isozyme of
3deoxy-D-arabino-heptulosonate-7-phosphate synthase insensitive to
feedback inhibition.
8. The method of claim 7, wherein the gene coding for the
3-deoxy-D-arabino-heptulosonate-7-phosphate synthase isozyme is an
aroF.sup.FBR gene.
9. The method of claim 8, wherein the aroF.sup.FBR is located on a
plasmid in the microbe.
10. The method of claim 1, wherein the aryl-aldehyde dehydrogenase
is purified from Neurospora crassa.
11. The method of claim 1, wherein the carbon source is
glucose.
12. A method of synthesizing vanillin from a carbon source
comprising:
a) converting the carbon source to vanillic acid with an E. coli
comprising recombinant genes encoding
3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase,
3-dehydroquinate synthase, 3-dehydroquinate synthase (aroB),
3-dehydroquinate dehydratase, 3-dehydroshikimate dehydrates and
catechol-O-methyltransfiemse; and
b) reducing vanillic acid to vanillin with arylaldehyde
dehydrogenase.
13. The method of claim 12, wherein the E. coli comprises a mutated
aroE locus encoding shikimate dehydrogenase, an aroB/aroZ cassette
inserted into a serA locus, and plasmid p5.97A.
14. The method of claim 12, wherein the aryl-aldehyde dehydrogenase
is purified from Neurospora crassa.
15. The method of claim 12, wherein the E. coli is KL7/pKL5.97A
identified by ATCC designation number 98859.
16. The method of claim 12, wherein the carbon source is
glucose.
17. A process for the production of vanillin comprising:
a) contacting a bioconversion mixture comprising a carbon source
with a microbe comprising a recombinant gene encoding a
3-dehydroshilcimate dehydratase and a recombinant gene encoding
catechol-O-methyltransferase, wherein the microbe converts the
carbon source to vanillic acid; and
b) contacting the vanillic acid with aryl-aldehyde dehydrogenase,
wherein the aryl-aldehyde dehydrogenase reduces the vanillic acid
to vanillin.
18. The process of claim 17, wherein the microbe is E. coli
comprising a mutated aroE locus, an aroB/aroZ cassette inserted
into the serA locus and plasmid pKL5.97A.
19. The process of claim 17, wherein the aryl-aldehyde
dehydrogenase is purified from Neurospora crassa.
20. The process of claim 17, wherein the microbe is E. coli
KL7/pKL5.97A identified by ATCC designation number 98859.
21. The process of claim 17, wherein the carbon source is
glucose.
22. The process of claim 17, wherein the bioconversion mixture is
maintained at a temperature of about 30.degree. C. to about
37.degree. C. and a pH of about 6.5 to about 7.5.
23. The process of claim 17, wherein the bioconversion mixture has
a dissolved oxygen concentration of about 5% to about 35% air
saturation.
24. The process of claim 17, wherein the steps are performed in a
fed-batch fermentor.
25. E. coli KL7/pKL5.97A identified by ATCC designation number
98859.
26. Plasmid pKL5.97A.
Description
FIELD OF THE INVENTION
The present invention is related to the bioconversion of a carbon
source to vanillin and more particularly, to methods of producing
vanillin from a carbon source by microbe-catalyzed conversion of
the carbon source to vanillic acid and enzyme-catalyzed reduction
of vanillic acid to produce vanillin.
BACKGROUND OF THE INVENTION
Natural vanillin is produced from glucovanillin (FIG. 1) when the
beans of the orchid Vanilla planifolia are submitted to a
multi-step curing process. Ranadive, A. S., In Spices, Herbs, and
Edible Fungi, Charalambous, G., Ed., Elsevier: Amsterdam, p. 517
(1994). Because of the extreme care that must be exercised during
vine cultivation, bean harvesting, and hand pollination of flowers,
natural vanillin can supply only 2.times.10.sup.4 kg/yr of the
world's 1.2.times.10.sup.7 kg/yr demand for vanillin. Clark, G. S.,
Perfum. Flavor. 15:45 (1990). This has resulted in substitution of
synthetic vanillin for natural vanilla in most flavoring
applications. Condensation of glyoxylic acid with benzene-derived
guaiacol (FIG. 1) is therefore currently the dominant route for
vanillin manufacture. Ranadive, A. S., In Spices, Herbs, and Edible
Fungi, Charalambous, G., Ed., Elsevier: Amsterdam, p. 517 (1994);
Clark, G. S., Perfum. Flavor. 15:45 (1990); Esposito, L. et al.,
Kirk-Othmer Encyclopedia of Chemical Technology, Fourth Ed.,
Kroschwitz, J. I.; Howe-Grant, M., Ed.; Wiley: New York, Vol.
24:812 (1997). Limited vanilla bean supplies have also led to
extensive research into the use of plant tissue culture and
microbes to convert ferulic acid (FIG. 1) into vanillin suitable
for labelling as a natural or nature-equivalent flavoring.
Falconnier, B. et al., J. Biotechnol. 37:123 (1994);
Lesage-Meessen, L. et al., J. Biotechnol. 50:107 (1996);
Lesage-Meessen, L. et al., Appl. Microbiol. Biotechnol. 47:393
(1997); Labuda, I. M. et al., U.S. Pat. No. 5,279,950 (1994);
Westcott, R. J. et al., Phytochemistry 35:135 (1994).
Vanillin is second only to aspartame in terms of market size for a
food additive. Vanilla extract derived from V. planifolia pods has
the advantage of being labeled as a natural flavoring. However, as
described above, only relative small volumes of vanilla flavoring
can be derived from V. planifolia cultivation. Synthesis of
vanillin from benzene-derived guaiacol is therefore the basis of
large-scale industrial manufacture of vanillin. This vanillin
however, can not be labeled as a natural flavoring and synthesis of
vanillin from benzene-derived guaiacol is not environmentally
benign. With respect to the ferulate-derived vanillin, although it
can be labeled as a natural flavoring, the microbes and cultured
plant cells used to process the ferulic acid give low titers of
vanillin (approximately 1 g/L). Falconnier, B. et al., J.
Biotechnol. 37:123 (1994); Lesage-Meessen, L. et al., J.
Biotechnol. 50:107 (1996); Lesage-Meessen, L. et al., Appl.
Microbiol. Biotechnol. 47:393 (1997); Labuda, I. M. et al., U.S.
Pat. No. 5,279,950 (1994); Westcott, R. J. et al.,
Phytochemistry35:135 (1994). A further problem is the availability
of ferulic acid; although corn fiber is rich in ferulic acid
esters, no process amenable to commercial scale isolation and
processing of this ferulic acid has been developed.
It would thus be desirable to provide a method for synthesizing
vanillin. It would further be desirable to provide a method for
synthesizing vanillin which is economically attractive. It would
also be desirable to provide a method for synthesizing vanillin
which is environmentally benign. It would further be desirable to
provide a method for synthesizing vanillin which utilizes an
abundant, renewable resource as the starting material.
SUMMARY OF THE INVENTION
A bioengineered synthesis scheme for the production of vanillin
from a carbon source is provided. In one embodiment, the
bioconversion methods of the present invention comprise the steps
of microbe-catalyzed conversion of a carbon source to vanillic acid
followed by enzyme-catalyzed reduction of vanillic acid to produce
vanillin. As shown in the synthesis scheme of FIG. 2, the
microbe-catalyzed conversion step of the present invention requires
five enzymes which are provided by a recombinant microbe. In a
preferred embodiment, the recombinant microbe is Escherichia coli
designed to cause dehydration of 3-dehydroshikimic acid and
regioselective methylation of the resulting protocatechuic acid.
The enzyme-catalyzed reduction step of the present invention
comprises the reduction of vanillic acid to vanillin by
aryl-aldehyde dehydrogenase. In a preferred embodiment, the
aryl-aldehyde dehydrogenase is purified from Neurospora crassa.
The biocatalytic synthesis of vanillin provided herein is
environmentally benign, economically attractive, and utilizes
abundant renewable sources, as starting materials.
Additional objects, advantages, and features of the present
invention will become apparent from the following description and
appended claims, taken in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
The various advantages of the present invention will become
apparent to one skilled in the art by reading the following
specification and subjoined claims and by referencing the following
drawings in which:
FIG. 1 is a schematic illustrating various synthesis schemes for
producing vanillin;
FIG. 2 is a schematic illustrating the synthesis scheme of the
present invention;
FIG. 3 is a graph showing the effect over time of extracellular
accumulation of various constituents on cells (g/L) and vanillate
(mM); and
FIG. 4 is a .sup.1 H NMR of vanillin synthesized from glucose.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
A bioengineered synthesis scheme for the production of vanillin
from a carbon source is provided herein. Methods of producing
vanillin from a carbon source based on the synthesis scheme are
also provided. In one embodiment, a method is provided wherein the
carbon source is converted to vanillic acid by a recombinant
microbe followed by the reduction of vanillic acid to vanillin by
aryl-aldehyde dehydrogenase. In a preferred embodiment, the
aryl-aldehyde dehydrogenase is isolated from Neurospora crassa.
Although microbe-catalyzed conversion of a carbon source to
vanillic acid followed by enzyme-catalyzed reduction of vanillic
acid to vanillin is described in detail herein, in an alternative
embodiment, a single recombinant microbe may is employed to convert
a carbon source to vanillic acid as well as reduce the vanillic
acid to vanillin, e.g., the vanillic acid-synthesizing microbe may
also express aryl-aldehyde dehydrogenase. This "single-microbe
conversion" may be carried out by any type of microbe sufficiently
engineered to produce the desired outcome, including, but not
limited to, E. coli, Klebsiella, Neurospora, Nocardia and
Saccharomyces.
In another embodiment, vanillic acid synthesized from a carbon
source by one microbe is reduced to vanillin by a second microbe,
wherein the second microbe expresses aryl-aldehyde dehydrogenase.
This "double-microbe conversion" may also be carried out by various
types of microbes sufficiently engineered to produce the desired
outcome. Neurospora and Nocardia are preferred as the second
microbe, as both are known to naturally express aryl-aldehyde
dehydrogenase.
In yet another embodiment, the microbe-catalyzed conversion of the
carbon source is to 3-dehydroshikimic acid followed by conversion
of the 3-dehydroshikimic acid to vanillin. In a further embodiment,
the microbe-catalyzed conversion of the carbon source is to
protocatechuic acid, followed by conversion of the protocatechuic
acid to vanillin. The conversion of 3-dehydroshikimic acid and/or
protocatechuic acid to vanillin may be carried out by a second
recombinant microbe engineered to provide such a conversion.
The bioconversion methods of the present invention are carried out
under conditions of time, temperature, pH, nutrient type and
concentration, aeration conditions, methionine supplementation, and
limited glucose concentrations, to provide maximal conversion of
the carbon source to vanillin. As described in detail in Specific
Example 1, in a preferred embodiment, a fed-batch fermentor is used
to convert the carbon source to vanillic acid, followed by organic
extraction of vanillic acid, e.g., acidification of the
fermentation broth and extraction with organic solvent. The
fed-batch fermentor process and organic extraction methods are also
known to those skilled in the art.
As used herein, the phrase "carbon source" is meant to include
biomass derived carbon sources including, but not limited to,
xylose, arabinose, glucose and the intermediates (e.g.,
dicarboxylic acids) in the Krebs cycle, either alone or in
combination. In a preferred embodiment, the carbon source is
glucose.
In one embodiment, the recombinant microbe E. coli is employed in
the methods of the present invention. In a preferred embodiment,
the E. coli comprises a mutated aroE locus and an aroB/aroZ
cassette inserted into the serA locus. This recombinant E. coli,
designated KL7, may further comprise a plasmid carrying an
aroF.sup.FBR insert, a serA insert and a P.sub.tac COMT loci. The
lack of aroE-encoded shikimate dehydrogenase results in synthesis
of 3-dehydroshikimic acid. It will be appreciated, however, that
the aroE locus mutation is not essential and is employed to ensure
sufficient 3-dehydroshikimic acid formation. The 3-dehydroshikimic
acid is converted into protocatechuic acid by genome-localized,
aroZ-encoded 3-dehydroshikimate dehydratase. Plasmid-localized
P.sub.tac COMT encodes catechol-O-methyltransferase for conversion
of protocatechuic acid into vanillic acid. In addition, the two
copies of aroB increase 3-dehydroquinate synthase activity to the
point where the enzyme no longer impedes carbon flow. Snell, K. et
al., J. Am. Chem. Soc. 118:5605 (1996).
In a preferred embodiment, the recombinant E. coil comprises
plasmid pKL5.97A carrying an aroF.sup.FBR insert, a serA insert and
two P.sub.tac COMT loci. The aroF.sup.FBR insert encodes a
3-deoxy-D-arabino-heptulosonic acid 7-phosphate synthase isozyme
insensitive to feedback inhibition which increases carbon flow into
the common pathway. Due to a mutation in the E. coli genomic serA
locus required for L-serine biosynthesis, growth in minimal salts
medium and plasmid maintenance follows from expression of
plasmid-localized serA. The serA insert thus allows microbial
growth in minimal salts medium, distinguishing the microbes
containing the plasmid from non-plasmid containing microbes.
In an alternative embodiment, the recombinant E. coli comprises
plasmid pKL5.96A which is identical to plasmid pKL5.97A except for
a single P.sub.tac COMT locus as compared to the double P.sub.tac
COMT loci in pKL5.97A.
The above-described preferred recombinant microbe of the present
invention, E. coli KL7/pKL5.97A, has been deposited with the
American Type Culture Collection (ATCC), 1080 University Boulevard,
Manassas, Va. 20110-2209, under the terms of the Budapest Treaty,
and has been accorded the ATCC designation number 98859. The
deposit will be maintained in the ATCC depository, which is a
public depository, for a period of 30 years, or 5 years after the
most recent request, or for the effective life of a patent,
whichever is longer, and will be replaced if the deposit becomes
depleted or nonviable during that period. Samples of the deposit
will become available to the public and all restrictions imposed on
access to the deposit will be removed upon grant of a patent on
this application.
The following table sets forth the five enzymes required for the
conversion of glucose to vanillic acid, the genes encoding same and
the origin of the genes in the exemplary recombinant microbes of
the present invention.
TABLE 1 Enzyme.sup..dagger. Gene (origin) a)
3-deoxy-D-arabino-heptulosonic aroF.sup.FBR (plasmid) acid
7-phosphate synthase b) 3-dehydroquinate synthase aroB (additional
copy inserted into genome) c) 3-dehydroquinate dehydratase aroD
(genomic) d) 3-dehydroshikimate dehydratase aroZ (inserted into
genome) e) catechol-O-methyltransferase P.sub.tac COMT (plasmid)
(COMT) .sup..dagger. Enzymes a)-e) correspond to a-e of FIG. 2.
Although E. coli is specifically described herein as the microbe
for carrying out the methods of the present invention, it will be
appreciated that any microorganism such as the common types cited
in the literature and known to those skilled in the art, may be
employed, provided the microorganism can be altered to effect the
desired conversion (e.g., carbon source to vanillic acid, carbon
source to 3-dehydroshikimic acid, carbon source to protocatechuic
acid, vanillic acid to vanillin, 3-dehydroshikimic acid to
vanillin, protocatechuic acid to vanillin, etc.) Thus, it is
envisaged that many types of fungi, bacteria and yeasts will work
in the methods of the present invention. Such microorganisms may be
developed, for example, through selection, mutation, and/or genetic
transformation processes with the characteristic and necessary
capability of converting one constituent of the synthesis scheme of
the present invention to another. Methods for such development are
well known to the skilled practitioner.
In order to carry out the bioconversion methods of the present
invention, a solution containing a carbon source is contacted with
the recombinant microbe to form a bioconversion mixture which is
maintained under appropriate conditions to promote the conversion
of the carbon source to the desired constituent, e.g., vanillic
acid. In a preferred embodiment, the bioconversion mixture is
maintained at a temperature of about 30.degree. C. to about
37.degree. C. and a pH of about 6.5 to about 7.5. It is preferred
that the bioconversion mixture also contain other substances
necessary to promote the viability of the recombinant microbes such
as mineral salts, buffers, cofactors, nutrient substances and the
like. Methionine (L, D and L-D mixtures) may also be added to the
bioconversion mixture. The bioconversion mixture is preferably
maintained in a steady state of dissolved oxygen concentration and
thus is kept under glucose limited conditions, wherein the rate of
glucose addition is determined by the level of dissolved oxygen
concentration. A preferred steady state over the course of
fermentation is about 100 to about 200 .mu.mol glucose or a
dissolved oxygen concentration of about 5% to about 35% air
saturation. The more general requirements for the maintenance of
viability of microorganisms are well known and specific
requirements for maintaining the viability of specific
microorganisms are also well known as documented in the literature,
or are otherwise easily determined by those skilled in the art. The
vanillic acid may then be recovered from the bioconversion mixture
by methods known in the art (e.g., organic extraction), and
contacted with aryl-aldehyde dehydrogenase to produce vanillin.
In order to more fully demonstrate the advantages arising from the
present invention, the following examples are set forth. It is to
be understood that the following is by way of example only and is
not intended as a limitation on the scope of the invention.
SPECIFIC EXAMPLE 1
Synthesis Of Vanillin From Glucose
I. Results
KL7/pKL5.26A and KL7/pKL5.97A were cultured for 48 h under
fed-batch fermentor conditions at 37.degree. C., pH 7.0, and
dissolved oxygen at 20% of saturation. Extracellular accumulation
(FIG. 3) of vanillic, isovanillic, protocatechuic, and
3-dehydroshikimic acids began in mid log phase of microbial growth.
3-Dehydroshikimic acid usually constituted 5-10 mol % of the total
product mixture indicating that the rates for its biosynthesis and
dehydration were nearly equal. However, the molar dominance of
protocatechuic acid (FIG. 3, Table 2) relative to vanillic acid
pointed to inadequate catechol-O-methyltransferase activity.
Although increasing the specific activity (Table 2) of
catechol-O-methyltransferase in KL7/pKL5.97A relative to
KL7/pKL5.26A had little impact on the concentrations (Table 2) of
synthesized vanillic acid, supplementation with L-methionine nearly
doubled the amount of vanillic acid synthesized by both
biocatalysts (Table 2). The 4-fold to 6-fold molar excess of
vanillic acid synthesized relative to isovanillic acid (Table 2)
conforms to the reported selectivity of
catechol-O-methyltransferase towards meta-hydroxyl group
methylation.
TABLE 2 Products formed after 48 h under fed-batch fermentor
conditions as a function of catechol-O-methyltransferase activity
and L-methionine supplementation. KL7/pKL5.26A.sup.a
KL7/pKL5.97A.sup.b L-methionine.sup.c - + - + COMT.sup.d 0.0060
0.0055 0.012 0.010 vanillic acid.sup.e 2.5 4.9 3.0 5.0 Isovanillic
acid.sup.e 0.4 1.3 0.6 1.2 protocatechuic acid.sup.e 9.7 7.1 12.9
10.5 3-dehydroshikimic acid.sup.e 0.9 1.0 1.0 1.8 .sup.a
aroF.sup.FBR P.sub.tac COMTserA .sup.b aroF.sup.FBR P.sub.tac
COMTP.sub.tac COMTserA .sup.c 0.4 g/L added every 6 h beginning at
12 h .sup.d specific activity: .mu.mol/min/mg .sup.e g/L
Aryl-aldehyde dehydrogenase (Gross, G. G. et al., Biochem. Biophy.
Res. Commun. 32:173 (1968); Gross, G. G. et al., Eur. J. Biochem.
8:413 (1969); Gross, G. G., Eur. J. Biochem. 31:585 (1972); Zenk,
M. H. et al., Recent Adv. Phytochem. 4:87 (1972)) in Neurospora
crassa mycelial extract was purified away from an unwanted
dehydrogenase which reduced vanillin to vanillyl alcohol. Vanillic,
protocatechuic, and isovanillic acids were extracted into EtOAc
after acidification of fermentor broth. A subsequent
reprecipitation step increased the vanillic acid/protocatechuic
acid ratio from 1:2 to 2.5:1 (mol/mol). The resulting aromatic
mixture was incubated with glucose 6-phosphate dehydrogenase (to
recycle NADP.sup.+) 25 and aryl-aldehyde dehydrogenase at
30.degree. C. and pH 8.0 using 0.07 equiv of NADP.sup.+ and 2 equiv
of ATP relative to vanillic acid. Reduction of vanillic acid to
vanillin (FIG. 2) proceeded in 92% yield in 7 h. Reduction of
protocatechuic acid was slower with a 33% yield of
protocatechualdehyde obtained after 7 h. Vanillin was extracted
from the enzymatic reduction with CH.sub.2 Cl.sub.2 leaving
protocatechualdehyde and protocatechuic acid in the aqueous phase.
Isovanillin at 10 mol % remained as the only contaminant.
Extraction of the fermentor broth, selective precipitation to
remove excess protocatechuic acid, aryl-aldehyde dehydrogenase
reduction, and the final CH.sub.2 Cl.sub.2 extraction led to a 66%
overall yield (mol/mol) for conversion of vanillic acid into
vanillin.
II. Materials and Methods
General. For .sup.1 H NMR quantitation of solute concentrations,
solutions were concentrated to dryness under reduced pressure,
concentrated to dryness one additional time from D.sub.2 O, and
then redissolved in D.sub.2 O containing a known concentration of
3-(trimethylsilyl)propionic-2,2,3,3-d.sub.4 acid (TSP) purchased
from Lancaster Synthesis Inc. Concentrations were determined by
comparison of integrals corresponding to each compound with the
integral corresponding to TSP (.delta.=0.00 ppm) in the .sup.1 H
NMR. All .sup.1 H NMR spectra were recorded on a Varian VXR-300
FT-NMR Spectrometer (300 MHz). HPLC analyses employed a Rainin
instrument, isocratic elution (17:2:1 H.sub.2 O/CH.sub.3
CN/CH.sub.3 CO.sub.2 H v/v), a C18 column (5 .mu.m, Rainin
Microsorb-MV.TM., 4.6.times.250 mm), and detection measured at 250
nm. Samples were quantitated by comparison of the peak area of each
component with a standard curve. Protein concentrations were
determined using the Bradford dye-binding procedure (Bradford, M.
M., Anal. Biochem. 72:248 (1976)) by comparison with a standard
curve prepared from bovine serum albumin. Protein assay solution
was purchased from Bio-Rad.
Enzyme Assays. A modification of the method of Reenila was used for
assay of catechol-O-methyltransferase activity. Reenila, I. et al.,
T. Pharmacol. Toxicol. 77:414 (1995). The cells were washed twice
with sodium phosphate (10 mM, pH 7.4) containing dithiothreitol
(0.5 mM) and resuspended in sodium phosphate (10 mM, pH 7.4)
containing dithiothreitol (0.5 mM). The cells were disrupted by two
passages through a French press (16000 psi). Cellular debris was
removed by centrifugation at 48000 g for 20 min. Cellular lysate
was diluted in sodium phosphate (10 mM, pH 7.4) containing
dithiothreitol (0.5 mM).
Two different solutions were prepared and incubated separately at
37.degree. C. for 3 min. The first solution (4 mL) contained sodium
phosphate (125 mM) pH 7.4, MgCl.sub.2 (6.25 mM),
S-adenosyl-L-methionine (0.75 mM), and protocatechuic acid (0.5
mM). The second solution (1 mL) consisted of the diluted lysate
containing catechol-O-methyltransferase. After the two solutions
were mixed (time=0), aliquots (0.5 mL) were removed at timed
intervals (1 min) and quenched with 40 .mu.L ice-cold 4 M
perchloric acid. Precipitated protein was removed by centrifugation
using a Beckman microfuge and components in the resulting
supernatant quantitated by HPLC. One unit of
catechol-O-methyltransferase activity was defined as the formation
of 1 .mu.mol of vanillic acid and isovanillic acid per min at
37.degree. C.
Aryl-aldehyde dehydrogenase assay solution (1 mL) containing
Tris-HCl (100 mM) pH 8.0, MgCl.sub.2 (10 mM), dithiothreitol (20
mM), NADPH (0.15 mM), ATP (20 mM), and benzoic acid (4 mM) was
incubated at 30.degree. C. After addition of solution containing
aryl-aldehyde dehydrogenase, benzoic acid reduction was monitored
at 340 nm using a Hewlett Packard 8452A UV-Vis spectrophotometer.
One unit of activity is defined as the loss of 1 .mu.mol of NADPH
per min at 30.degree. C.
Purification of Aryl-aldehyde Dehydrogenase. Whatman
(diethylaminoethyl)cellulose (DE52) and Amicon Dye Matrex Red A
gels were used during the purification. Buffers included buffer A,
Tris-HCl (100 mM) and L-cysteine (10 mM), pH 7.6; buffer B,
Tris-HCl (50 mM), EDTA (1 mM), DTT (1 mM), and PMSF (0.4 mM), pH
7.6; buffer C, Tris-HCl (50 mM), EDTA (1 mM), DTT (1 mM), PMSF (0.4
mM), and KCl (400 mM), pH 7.6; buffer D, Tris-HCl (20 mM), EDTA
(0.4 mM), DTT (0.4 mM), and PMSF (0.15 mM), pH 7.5; and buffer E,
Tris-HCl (20 mM), EDTA (0.4 mM), DTT (0.4 mM), PMSF (0.15 mM), and
KCl (2.5 M), pH 7.5. All protein purification manipulations were
carried out at 4.degree. C. Protein solution was concentrated by
ultrafiltration (PM-10 Diaflo membranes from Amicon).
All medium for cultivation of Neurospora crassa SY 7A was prepared
in distilled, deionized water. N. crassa SY 7A was obtained from
the American Type Culture Collection, ATCC designation number
24740. The solid growth medium (1 L) contained sucrose (20 g),
sodium citrate dihydrate (2.5 g), KH.sub.2 PO.sub.4 (5.0 g),
NH.sub.4 NO.sub.3 (2.0 g), CaCl.sub.2.OR left.2H.sub.2 O (0.1 g),
MgSO.sub.4 (0.1 g), biotin (5.0 .mu.g), and trace elements
including citric acid monohydrate (5.0 mg), ZnSO.sub.4.OR
left.7H.sub.2 O (5.0 mg), Fe(NH.sub.4).sub.2 (SO.sub.4).sub.2.OR
left.6H.sub.2 O (1.0 mg), CuSO.sub.4.OR left.5H.sub.2 O (0.25 mg),
MnSO.sub.4.OR left.H.sub.2 O (0.05 mg), H.sub.3 BO.sub.3 (0.05 mg),
Na.sub.2 MoO.sub.4.OR left.2H.sub.2 O (0.05 mg). Difco agar was
added to the medium solution at a concentration of 2% (w/v). The
liquid growth medium differed from solid growth medium only in the
addition of Difco yeast extract (2.0 g/L) and sodium salicylate
(1.6 g/L). N. crassa SY 7A was grown on solid growth medium at
24.degree. C. for 7 days and a mixture of mycelium and spores was
obtained. After suspension in sterilized water, the mixture of
mycelium and spores was filtered through sterilized glass wool. The
resulting spore suspension was stored at 4.degree. C. Fresh spores
stored at 4.degree. C. for less than 2 weeks were inoculated into 2
L liquid growth medium in a 4 L Erlenmeyer flask to give a final
concentration of 2.5.times.10.sup.6 spores/L. Kirk, T. K. et al.,
Arch. Microbiol. 117:277 (1978). After culturing at rt for 60 h,
the mycelium was harvested by filtration and frozen at -20.degree.
C.
Yields and specific activities at each step of the purification of
aryl-aldehyde dehydrogenase are summarized in Table 3. The specific
activity of aryl-aldehyde dehydrogenase could not be determined in
crude mycelial extract because of the presence of other
dehydrogenase activities. The frozen mycelium (400 g, wet weight)
was thawed in 900 mL buffer A and then disrupted with a Waring
blender. The debris was removed by centrifugation at 40000 g for 30
min followed by concentration of the supematant to 200 mL. After
dialysis against buffer B (3.times.), the mycelium extract was
applied to a DEAE column (5.times.23 cm) equilibrated with buffer
B. The column was washed with 500 mL of buffer B followed by
elution with a linear gradient (1.5 L+1.5 L, buffer B-buffer C).
Fractions containing aryl-aldehyde dehydrogenase were combined and
concentrated to 30 mL. After dialysis against buffer D (3.times.),
The protein was loaded on a RedA column (2.5.times.8 cm)
equilibrated with buffer D. The column was washed with 200 mL
buffer D and eluted with a linear gradient (150 mL+150 mL, buffer
D/buffer E). Active fractions were concentrated, quick frozen in
liquid nitrogen, and stored at -80.degree. C.
TABLE 3 Purification of aryl-aldehyde dehydrogenase from N. crassa
SY 7A. total units.sup.a specific activity.sup.b x-fold
purification yield crude lysate -- -- -- -- DEAE 58 0.072 1 100%
RedA 55 0.52 7 96% .sup.a 1 unit = 1 .mu.mol NADH oxidized/min.
.sup.b.mu.mol/min/mg
Vanillic Acid Synthesis. Fermentations employed a 2.0 L capacity
Biostat MD B-Braun fermentor connected to a DCU system and a Compaq
computer equipped with B-Braun MFCS software for data acquisition
and automatic process monitoring. The temperature, pH and glucose
feeding were controlled with a PID controller. The temperature was
maintained at 37.degree. C. pH was maintained at 7.0 by addition of
concentrated NH.sub.4 OH or 2 N H.sub.2 SO.sub.4. Dissolved oxygen
(D.O.) was measured using a Braun polarographic probe. D.O. was
maintained at 20% air saturation over the entire course of the
fermentation. Antifoam (Sigma 204) was added manually as
needed.
All medium was prepared in distilled, deionized water. LB medium (1
L) contained Bacto tryptone (10 g), Bacto yeast extract (5 g), and
NaCl (10 g). Fermentation medium (1 L) contained K.sub.2 HPO.sub.4
(7.5 g), ammonium iron(III) citrate (0.3 g), citric acid
monohydrate (2.1 g), and concentrated H.sub.2 SO.sub.4 (1.2 mL).
The culture medium was adjusted to pH 7 by addition of concentrated
NH.sub.4 OH before autoclaving. The following supplements were
added immediately prior to initiation of the fermentation:
D-glucose (20 g), MgSO.sub.4 (0.24 g), aromatic amino acids
including phenylalanine (0.7 g), tyrosine (0.7 g), and tryptophan
(0.35 g), aromatic vitamins including p-aminobenzoic acid (0.01 g),
2,3-dihydroxybenzoic acid (0.01 g), and p-hydroxybenzoic acid (0.01
g), and trace minerals including (NH.sub.4).sub.6 (Mo.sub.7
O.sub.24).OR left.4H.sub.2 O (0.0037 g), ZnSO.sub.4.OR
left.7H.sub.2 O (0.0029 g), H.sub.3 BO.sub.3 (0.0247 g),
CuSO.sub.4.OR left.5H.sub.2 O (0.0025 g), and MnCl.sub.2.OR
left.4H.sub.2 O (0.0158 g). D-Glucose, MgSO.sub.4, and aromatic
amino acids were autoclaved while aromatic vitamins and trace
minerals were sterilized through 0.22 .mu.m membranes prior to
addition to the medium. Antibiotics were added where appropriate to
the following final concentrations: chloramphenicol (Cm), 20
.mu.g/mL; ampicillin (Ap), 50 .mu.g/mL. Solid medium was prepared
by addition of 1.5% (w/v) Difco agar to medium solution.
Inoculants were grown in 100 mL LB medium (enriched with 2 g
glucose) containing the appropriate antibiotic for 12 h at
37.degree. C. with agitation at 250 rpm and then transferred to the
fermentor. The initial glucose concentration in the fermentation
medium was 20 g/L. L-Methionine supplementation, when employed,
consisted of addition of a filter-sterilized solution containing
0.4 g of this amino acid in timed intervals (6 h) starting at 12 h
after initiation of a fermentor run. Three different methods were
used to maintain dissolved oxygen (D.O.) levels at 20% air
saturation during each 48 h fermentor run. The dissolved oxygen
concentration was first maintained by increasing the impeller
speed. Approximately 8 h was required for the impeller speed to
increase from 50 rpm to the preset maximum value of 900 rpm. The
mass flow controller then maintained D.O. levels at 20% saturation
at constant impeller speed by increasing the airflow rate over
approximately 2 h from 0.06 L/L/min to a preset maximum of 1.0
L/L/min. At constant impeller speed and constant airflow rate, D.O.
levels were maintained at 20% saturation for the remainder of the
fermentation by oxygen sensor-controlled glucose feeding. At the
beginning of this stage, dissolved oxygen levels fell below 20%
saturation due to residual initial glucose in the medium. This
lasted for approximately 1 h before glucose (60% w/v) feeding
started. The PID control parameters were set to 0.0 (off for the
derivative control (T.sub.D), 999.9 s (minimum control action) for
the integral control (T.sub.l), and 950.0% for the proportional
band (X.sub.p).
Samples (6 mL) of fermentation broth were taken at 6 h intervals. A
portion (1 mL) was used to determine cell densities by measurement
of absorption at 600 nm (OD.sub.600). Dry cell weight (g/L) was
obtained using a conversion coefficient of 0.43 g/OD.sub.600 L. The
remaining 5 mL of each fermentation froth sample was centrifuged
using a Beckman microfuge and analyzed by HPLC. A separate aliquot
(25 mL) of fermentation broth was taken and centrifuged at 12 h and
36 h for assay of catechol-O-methyltransferase activity. Since
stable catechol-O-methyltransferase activity was observed over the
course of the fermentation, reported catechol-O-methyltransferase
activity (Table 1) is the average of 12 h and 36 h specific
activities. After 48 h, cells were removed by centrifugation at
16000 g for 10 min and the supernatant stored at 4.degree. C.
Reduction of VanillicAcid. Fermentation broth (100 mL) was
acidified to pH 3.1 using concentrated HCl and the resulting
precipitated protein was removed by centrifugation at 16000 g for
10 min. After extraction of the supernatant with EtOAc (3.times.),
the solvent was removed under reduced pressure. The resulting solid
was dissolved in 12 mL of water adjusted to pH 7.5 by NaOH (10 N)
addition. Subsequent dropwise addition of concentrated sulfuric
acid acidified the solution to pH 1.8 and resulted in precipitation
of a solid which was filtered and dried. The collected precipitate
was dissolved in a solution (100 mL) containing Tris-HCl (200 mM),
pH 8.0, MgCl.sub.2 (100 mM), DTT (10 mM), ATP (60 mM), NADP.sup.+
(2 mM), glucose 6-phosphate (60 mM), 2,000 units of glucose
6-phosphate dehydrogenase and 200 unit of the partially purified
aryl-aldehyde dehydrogenase. Reduction proceeded at 30.degree. C.
and was monitored by HPLC. After 7 h reaction, 92% (mol/mol) of the
starting vanillic acid and 34% (mol/mol) of the protocatechuic acid
had been reduced. The reaction mixture was extracted with 100 mL
CH.sub.2 Cl.sub.2 (3.times.). The combined organic extracts were
washed one time with equal volume of water. Concentration afforded
a powder consisting of (FIG. 4) vanillin (0.30 g) and isovanillin
(0.03 g).
SPECIFIC EXAMPLE 2
Commercial Applications
For large-scale vanillin synthesis, an intact microbe (as opposed
to cell-free enzyme systems) to reduce vanillic acid is preferred.
However, it should be appreciated that irrespective of the strategy
employed, improved protocatechuic acid methylation will be
essential. The lack of significantly improved protocatechuic acid
methylation with increased catechol-O-methyltransferase activity
and the improvement in methylation observed with L-methionine
supplementation suggest that cosubstrate S-adenosylmethionine
availability and/or feedback inhibition (Coward, J. et al.,
Biochemistry 12:2291 (1973)) may be limiting in vivo
methyltransferase activity. Improving regioselectivity for
protocatechuic acid meta-oxygen methylation using a different
isozyme of widely distributed (Gross, G. G. et al., Biochem.
Biophy. Res. Commun. 32:173 (1968); Gross, G. G. et al., Eur. J.
Biochem. 8:413 (1969); Gross, G. G., Eur. J. Biochem. 31:585
(1972); Zenk, M. H. et al., Recent Adv. Phytochem. 4:87 (1972))
catechol-O-methyltransferase is also advantageous. In addition, a
vanillate-synthesizing microbe designed with a protocatechuic acid
uptake system so that protocatechuic acid escaping into the culture
supernatant can be transported back into the cytoplasm for
methylation, would also be desirable.
Biocatalytic synthesis of vanillin from a carbon source such as
glucose has a number of advantages relative to other biocatalytic
vanillin syntheses. Coniferol, formed during phenylpropanoid
biosynthesis, is converted into coniferin by a glucosyltransferase
in Vanilla planifolia. Ranadive, A. S., In Spices, Herbs, and
Edible Fungi, Charalambous, G., Ed., Elsevier: Amsterdam, p.517
(1994). Coniferin is then transformed into glucovanillin which is
finally hydrolyzed by a .beta.-glucosidase. Ranadive, A. S., In
Spices, Herbs, and Edible Fungi, Charalambous, G., Ed., Elsevier:
Amsterdam, p. 517 (1994). Synthesis of vanillin via
3-dehydroshikimic, protocatechuic, and vanillic acids as taught by
the present invention, circumvents phenylpropanoid biosynthesis and
glucosylation/deglucosylation reactions. This substantially reduces
the number of enzymes required to synthesize vanillin.
Biocatalytic synthesis of vanillin from a carbon source such as
glucose also has advantages relative to synthetic vanillin
manufacture. Esposito, L. et al., Kirk-Othmer Encyclopedia of
Chemical Technology, Fourth Ed., Kroschwitz, J. l.; Howe-Grant, M.,
Ed.; Wiley: New York, Vol. 24:812 (1997). Phenol and guaiacol are
toxic and are derived from carcinogenic benzene. Lewis, R. J. Sr.,
Hazardous Chemicals Desk Reference, Third Edition, Van Nostrand
Reinhold: New York (1993). The nontoxic 3-dehydroshikimic,
protocatechuic, and vanillic acids of the methods of the present
invention are derived from innocuous glucose. Corrosive H.sub.2
O.sub.2 used for oxidation of phenol into catechol requires special
handling precautions (Campbell, C. J. et al., Sci. Am. 278(3):78
(1998)) while biocatalytically synthesized vanillin derives its
oxygen atoms from the oxygen atoms of glucose. Dimethyl sulfate, a
carcinogen, (Campbell, C. J. et al., Sci. Am. 278(3):78 (1998)) has
historically been used to methylate catechol. Protocatechuic acid
methylation employs S-adenosylmethionine generated and consumed
intracellularly. Finally, synthetic vanillin manufacture is based
on use of nonrenewable petroleum whereas glucose is derived from
abundant, renewable starch. This difference in feedstock
utilization is important given projected fierce international
competition as global petroleum production diminishes. Campbell, C.
J. et al., Sci. Am. 278(3):78 (1998).
The foregoing discussion discloses and describes merely exemplary
embodiments of the present invention. One skilled in the art will
readily recognize from such discussion, and from the accompanying
drawings and claims, that various changes, modifications and
variations can be made therein without departing from the spirit
and scope of the invention as defined in the following claims.
Patent and literature references cited herein are incorporated by
reference as if fully set forth.
* * * * *